Method for producing recombinant trypsin

ABSTRACT

The present invention concerns a method for producing recombinant trypsin from porcine pancreas in  Pichia pastoris  which is soluble and secreted into the culture medium, whereby expression at pH 3.0-4.0 substantially prevents activation of trypsinogen to β-trypsin and autolysis of β-trypsin by α-trypsin into ε-trypsin and from there into inactive peptides.

[0001] The invention concerns a method for the recombinant production oftrypsin. For this purpose a trypsinogen with a shortened propeptidesequence is preferably expressed in a recombinant host cell and secretedinto the culture medium in an uncleaved form. Subsequently thepropeptide sequence is cleaved in a controlled manner to form activetrypsin.

[0002] Trypsin is a serine protease which catalyses a hydrolyticcleavage of peptides at the carboxyl group of the basic amino acidsarginine and lysine (Keil B., 1971). Trypsin from bovine pancreas wasone of the first proteolytic enzymes that could be used in a pure formand in adequate quantities for exact chemical and enzymatic studies(Northrop et al., 1948). It was subsequently also possible to isolateproteases that can be allocated to the trypsin family from other highervertebrates (pig—Charles et al. 1963/sheep—Bricteux-Gregoire et al.(1966); Travis (1968)/turkey—Ryan (1965)/humans—Travis et al. (1969) andothers). At this time the first enzymes belonging to the trypsin familywere also isolated from two species of streptomyces (Morihara andTsuzuki (1968); Trop and Birk (1968); Wählby and Engström (1968); Wählby(1968; Jurasek et al. (1969)).

[0003] The enzyme is synthesized in the pancreas cells of vertebrates asan inactive precursor, trypsinogen, and subsequently converted into theactive form by cleavage of the propeptide (Northrop et al. (1948),Desnuelle (1959)). The first trypsinogen molecules were activatednaturally by the enteropeptidase enterokinase which hydrolyses thepeptide bond between (Asp₄)-Lys-

-Ile which cleaves off the propeptide (Keil (1971)). The recognitionsequence of the enterokinase (Asp₄)-Lys is accordingly located directlyat the C-terminus of the propeptide in almost all previously knowntrypsinogen molecules (Light et al. (1980)). The activation process canalso proceed autocatalytically at physiological pH values since a lysineis located on the C-terminal side of the enterokinase recognitionsequence and hence the Lys-

-Ile peptide bond can also be hydrolyzed by trypsin (Light et al.(1980)).

[0004] Trypsin has always been an interesting protease forbiotechnological applications due to its ready availability from variousmammals, high specificity (only cleaves at the C-terminal side of lysineor arginine) together with high specific activity (˜150 U/mg) and itsgood storage stability. Trypsin is mainly used for the tryptic cleavageof peptides into small sections for sequencing, for detaching adherentcells from coated cell culture dishes and for cleaving fusion proteinsinto the target peptide and the fusion component, for activatingpropeptides (e.g. trypsinogen to trypsin) and for the recombinantproduction of peptide hormones (e.g. proinsulin to insulin, cf. WO99/10503). Trypsin is also a component of some pharmaceuticalpreparations (ointments, dragées and aerosols for inhalation (“RoteListe”, 1997; The United States Pharmacopeia, The National Formulary,USP23-NF18, 1995)). Since the use of enzymes from animal sources is nolonger permitted in many cases (potential contamination with infectiousagents), recombinant trypsin molecules for the desired biotechnologicalapplications have to be provided from microbial hosts.

[0005] There are several methods for the recombinant production oftrypsin from various organisms.

[0006] Graf, L. et al (1987 and 1988) describe the expression andsecretion of rat trypsin and trypsinogen mutants in E. coli. In order tosecrete the trypsinogen molecules into the periplasm of E. coli thenative trypsinogen signal sequence is replaced by the signal sequence ofthe bacterial alkaline phosphatase (phoA). The secreted inactivetrypsinogen molecules are isolated from the periplasm and activated byenzymatic cleavage using purified enterokinase.

[0007] Vasquez, J. R. et al. (1989) describe the expression andsecretion of anionic rat trypsin and trypsin mutants in E. coli. Inorder to express and secrete the active trypsin molecules into theperiplasm of E. coli, the native trypsinogen prepro segment (signalsequence and activation peptide) is replaced by the signal sequence ofthe bacterial alkaline phosphatase (phoA) and the phoA promoter that canbe regulated by phosphate is used. Active trypsin is isolated from theperiplasm. However, the yield is very low (ca. 1 mg/l).

[0008] Higaki, J. N. et al. (1989) describe the expression and secretionof trypsin and trypsin mutants into the periplasm of E. coli using thetac promoter and the S.typhimurium hisj signal sequence. The yield ofactive trypsin is ca. 0.3 mg/l. The volume yield of active anionic rattrypsin can be increased to about 50 mg/l by high cell densityfermentation (Yee, L. and Blanch, H. W., (1993)). However, the authorsrefer to problems in the expression and secretion of active trypsin inE. coli. Enzymatically active trypsin is formed in the periplasm of E.coli after cleavage of the signal sequence and native trypsin proteinfolding to form 6 disulfide bridges. The formation of active trypsin istoxic for the cell since active trypsin hydrolyses the periplasmatic E.coli proteins which lyses the cells. Moreover the protein folding oftrypsin and in particular the correct native formation of the 6disulfide bridges appears to be impeded in the periplasm of E. coli. Thesystem is not suitable for the isolation of relatively large amounts oftrypsin (>10 mg; Willett, W. S. et al., (1995)).

[0009] In order to produce larger amounts of trypsin (50-100 mg) forX-ray crystallographic investigations, an inactive trypsinogen precursoris produced in yeast under the control of a regulatable ADH/GAPDHpromoter and secreted by fusion with the yeast α factor leader sequence.The expression product secreted into the medium is convertedquantitatively into trypsin in vitro by means of enterokinase. The yieldis 10-15 mg/l (Hedstrom, L. et al. (1992)).

[0010] DNA sequences are described in EP 0 597 681 which code for maturebovine trypsin and bovine trypsinogen with an initial methionineresidue. In addition the expression in E. coli is described but thestrategy of how active trypsin is formed in E. coli is not explained.

[0011] A method for producing trypsin from porcine pancreas or aderivative thereof in Aspergillus by a recombinant method is describedin WO 97/00316. A vector is used for transformation which codes fortrypsinogen or a derivative thereof which is fused at the N-terminus toa functional signal peptide. However, yeast cultures achieve higherbiomass concentrations compared to Aspergillus cultures and growconsiderably more rapidly and thus the specific expression yield peryeast cell can be less than that for Aspergillus cells in order toachieve yields that are required for an economic expression method.

[0012] A method for the recombinant production of a zymogenic precursorof proteases which contains an autocatalytic cleavage site that does notoccur naturally is described in WO 99/10503. The method comprises anexpression of the zymogenic precursor in E. coli in the form ofinclusion bodies with subsequent purification and renaturation underconditions where the protease part of the zymogenic precursor is formedin its natural conformation and the cleavage of the renatured zymogenicprecursor then occurs autocatalytically. A disadvantage of this methodare the large losses that often occur during renaturation and the largevolumes that are required for an industrial production.

[0013] The recombinant production of trypsin analogues in Pichiapastoris is described in WO 00/17332. A vector is used for thetransformation which codes for a trypsinogen analogue (derivative ofbovine trypsinogen) in which the amino acid lysine at the C-terminus ofthe propeptide was exchanged by mutation for another amino acid (apartfrom arginine or lysine) and which is fused N-terminally to a functionalsignal peptide. In this method the trypsin analogues are secreted intothe medium in a soluble form and as a result of the incorporatedmutation are also not activated and further degraded by undesiredautocatalysis even at relatively high pH values of the fermentationprocess. An aminopeptidase is then used for activation. However, adisadvantage of this method is the need to remove the additionalaminopeptidase which may have disadvantageous side activities for thesubsequent use of the trypsin in the final process.

[0014] The expression of naturally occurring trypsinogen genes in Pichiapastoris is described in WO 01/55429. In order to avoid autocatalyticactivation the fermentation is carried out in the example in the lessacid pH range at ca. 6. It is thus outside the optimal pH range whichprevents autocatalytic activation during longer run times in theexpression phase. Other disadvantages of the method are the trypsinogengenes that are not codon-optimized for expression in Pichia pastoris andthe associated low expression yields.

[0015] The object of the invention is to provide a method for therecombinant production of trypsin in which the disadvantages of theprior art are at least partially eliminated and which allows activetrypsin to be obtained in a simple manner in a high yield and activity.In particular it should be possible to isolate trypsinogen as anintermediate product in a soluble form from the culture medium of theexpression host which should not be subject to any substantial degree ofautocatalytic activation during the fermentation and purification.Furthermore it should be possible to subsequently activate thetrypsinogen autocatalytically and/or by adding recombinant trypsin.

[0016] The object according to the invention is achieved by a method forthe recombinant production of trypsin comprising the steps:

[0017] a) transforming a host cell with a recombinant nucleic acid whichcodes for a trypsinogen with an enterokinase recognition site in thepropeptide sequence in a secretable form, preferably fused with a signalpeptide that mediates secretion,

[0018] b) culturing the host cell under conditions which enable anexpression of the recombinant nucleic acid and a secretion of theexpression product into the culture medium, the conditions beingselected such that an autocatalytic cleavage of the propeptide sequenceis at least substantially prevented,

[0019] c) isolating the expression product from the culture medium,

[0020] d) cleaving the propeptide sequence to form active trypsin and

[0021] e) optionally separating non-cleaved trypsinogen molecules fromactive trypsin.

[0022] The host cell used in the method according to the invention canbe a eukaryotic or prokaryotic host cell like those known from the priorart. The host cell is preferably a eukaryotic cell, particularlypreferably a fungal cell and most preferably a yeast cell. Suitableexamples of yeast cells are Pichia pastoris, Hansenula polymorpha,Saccharomyces cerevisiae, Schizosaccharomyces pombae wheremethylotrophic yeasts such as Pichia pastoris or Hansenula polymorphaand in particular Pichia pastoris are preferably used.

[0023] It is expedient to use a host cell that is able to express therecombinant nucleic acid and to secrete the expression product into theculture medium under conditions where an autocatalytic cleavage of thepropeptide sequence is at least substantially and preferably essentiallyquantitatively prevented. Such conditions advantageously comprise anacidic pH value of the culture medium, particularly preferably a pHvalue in the range between 3 and 4. Trypsinogen is stable at acidic pHvalues and especially at pH values of up to 4 whereas trypsin isautocatalytically activated in a neutral or alkaline medium (Keil, B.(1971)).

[0024] The culture under suitable conditions that occurs in the methodaccording to the invention prevents a premature activation of therecombinant trypsinogen secreted by the host cell which can alsosubstantially prevent further degradation of the resulting trypsin to asfar as inactive peptides. The stability of the recombinant trypsinogenunder the culture conditions is important since the expression should beaccompanied by a secretion. In this connection secretion from the hostcell is understood as the discharge of trypsinogen from the cytoplasmthrough the cell membrane into the culture medium. This usually occursby N-terminal fusion of the trypsinogen with a functional signalpeptide. Examples of suitable signal peptides are known signal peptidesfrom yeast and especially preferably the signal peptide of the α factorfrom Saccharomyces cerevisiae. However, it is of course also possible touse other signal peptides e.g. the signal peptide which naturallycontrols the secretion of trypsinogen.

[0025] In order to improve the expression yield of the recombinanttrypsin, a recombinant nucleic acid with an optimized codon usage forthe respective host cell is preferably used. Accordingly it isadvantageous to use a nucleic acid for a yeast cell which has anoptimized codon usage for yeast. Such nucleic acids having an optimizedcodon usage can for example be produced by synthesizing individualoligonucleotides in parallel and subsequently combining them.

[0026] The method according to the invention is basically suitable forproducing any types of recombinant trypsin provided the respectivetrypsinogen that is secreted as an expression product by the host cellis essentially stable under the culture conditions. The method ispreferably used to produce trypsin from vertebrates, in particular frommammals such as pigs, sheep or humans. Porcine trypsin is particularlypreferably produced.

[0027] Furthermore it is preferred that a recombinant nucleic acid isused which codes for a trypsinogen with a shortened propeptide sequence.Trypsinogen is understood as a protein which, although it has formed asubstantially correct protein structure, has no or only a very lowproteolytic activity compared to active trypsin which is advantageouslyat least 5-fold and particularly preferably at least 10-fold less thanthe proteolytic activity of the active form. The natural length of thepropeptide part, e.g. 25 amino acids in the case of a porcinetrypsinogen, is shortened preferably down to a propeptide sequence whichonly consists of a recognition sequence for an enteropeptidase e.g.enterokinase e.g. an amino acid sequence Asp-Asp-Asp-Asp-Lys. A fewadditional amino acids that are due to the cloning, e.g. up to 5 aminoacids, can be optionally attached to the N-terminus of the enterokinaserecognition sequence.

[0028] It was found that the expression of a shortened recombinanttrypsinogen according to the invention containing the amino acidsGlu-Phe attached due to the cloning to the N-terminus of theenterokinase recognition site results in significantly higher yieldsthan is the case for the expression of natural trypsinogen in the methodaccording to the invention. Hence in a particularly preferred embodimentof the method according to the invention a recombinant nucleic acidhaving the nucleotide sequence shown in SEQ ID NO.22 is used which codesfor a porcine trypsinogen with a shortened propeptide sequence.

[0029] In the method according to the invention the expression of therecombinant trypsinogen is preferably controlled by an inducibleexpression control sequence. Hence the growth of the host cell can takeplace before induction of expression under conditions which arefavourable or optimal for the growth of the host cell. Thus for examplegrowth can take place at pH 5-7, in particular pH 5-6 up to apredetermined optical density during which the regulatable expressioncontrol sequence is repressed. Expression can then be induced bychanging the temperature and/or by adding an inducer. An example of apreferred expression control sequence is the AOX 1—promoter from Pichiapastoris that can be induced by methanol which is particularly suitablefor inducible expression in methylotrophic yeasts. The cultureconditions are advantageously changed before induction of the expressioncontrol sequence in such a manner that for example by changing the pH toa range of 3-4 the trypsinogen formed by expression of the recombinantnucleic acid accumulates in an intact form in the culture medium.

[0030] The culture conditions in the method according to the inventionare selected such that autocatalytic cleavage of the propeptide sequenceis substantially prevented. After isolating the expression product fromthe culture medium, the propeptide sequence can be cleaved off undercontrolled conditions. This cleavage can for example be carried out byadding recombinant trypsin or/and by autocatalytic cleavage. In thisconnection autocatalytic cleavage is understood as the self-activationof recombinant trypsinogen which may be optionally accelerated by addingsmall amounts of recombinant trypsin but without adding a foreignprotein. In this manner it is not necessary to use an additional foreignprotein which is usually derived from animal raw material sources andhas to be subsequently removed again which could cause undesiredcleavages in the subsequent application. The autocatalytic activationpreferably occurs at a pH in the range of 7-8. This ensures that thetrypsinogen formed during the expression can be firstly highly purifiedin a strongly acidic range and the activation can be specificallystarted by rebuffering preferably in the presence of small amounts e.g.20 mM CaCl₂ to a neutral to weakly basic pH range. The activation can bestopped by changing the pH again to a strongly acidic range. Trypsin canfor example be purified by chromatography on ion exchanger material suchas SP-Sepharose®XL or benzamidine-Sepharose.

[0031] In order to purify the expression product from the culturemedium, the culture supernatant can be firstly separated from the cellsas described in example 6.1. The subsequent purification comprising anautocatalytic activation of the trypsinogen is preferably carried out bysuitable chromatographic purification procedures. In a particularlypreferred embodiment a chromatography is carried out as described inexample 6.2. without prior separation of the cells in which a buffercontaining calcium ions preferably at a final concentration of 1-30 mMcalcium is added to the culture medium which still contains the cells.Subsequently steps (c), (d) and optionally (e) of the method accordingto the invention are carried out for example using suitablechromatographic purification procedures and a rebuffering step for theautocatalytic cleavage of the expression product.

[0032] Hence a particularly preferred embodiment of the method accordingto the invention is characterized by:

[0033] a) transforming a host cell with a recombinant nucleic acid whichcodes for the zymogenic precursor trypsinogen from the pig which isfused with a signal peptide sequence and contains a propeptide which isshortened down to the enterokinase recognition sequence, where thiscleavage site is cleaved by recombinant trypsin or is autocatalyticallycleaved under certain buffer conditions and the shortened trypsinogencan be cleaved to form active trypsin in this process,

[0034] b) culturing the host cell during the expression phase at anacidic pH, preferably pH 3-4 so that the shortened trypsinogen ispresent in a soluble form and secreted into the culture medium but theautocatalytic activation is substantially prevented,

[0035] c) isolating the shortened recombinant trypsinogen from theculture supernatant and activating it under conditions which allow aneffective cleavage of the shortened trypsinogen by recombinant trypsinor by autocatalytic cleavage and

[0036] d) optionally separating non-cleaved trypsinogen molecules fromactive trypsin.

[0037] The shortening of the propeptide described in this invention hasa positive effect especially on expression and autocatalytic activation.Furthermore after the autocatalytic activation, the undesired furtherautolysis increases significantly at a pH of >4.0 during the growthphase and expression phase of the culture.

[0038] An additional increase in the expression yield in the methodaccording to the invention can be achieved by transforming the host cellwith several vectors each of which contains a recombinant nucleic acidas stated above where the vectors contain different selection markerse.g. Zeocin and G418. Culture under multiple selection conditionssurprisingly allows the expression yield of recombinant trypsinogen tobe increased considerably further.

[0039] Another subject matter of the invention is a recombinant nucleicacid which codes for a trypsinogen having an enterokinase recognitionsite in the propeptide sequence where the propeptide sequence isshortened relative to the natural propeptide sequence and is preferablyfused with a signal peptide sequence. The shortened propeptide sequenceaccording to the invention preferably consists of an enterokinaserecognition site having the amino acid sequence Asp-Asp-Asp-Asp-Lys andoptionally up to 5 additional amino acids located arranged N-terminallythereof. The nucleic acid according to the invention particularlypreferably has the nucleotide sequence shown in SEQ ID NO.22.

[0040] The nucleic acid according to the invention is preferably inoperative linkage with a regulatable expression control sequence whichis for example a suitable expression control sequence for geneexpression in yeast cells such as the AOX1 promoter from Pichiapastoris.

[0041] The invention also concerns a recombinant vector which containsat least one copy of a recombinant nucleic acid as stated above. Thevector is preferably a vector that is suitable for gene expression inyeast cells. Examples of such vectors are described in Sambrook et al.,Molecular cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.

[0042] In addition to the recombinant nucleic acid, the vector containsother suitable genetic elements for the respective intended use and inparticular a selection marker gene. Vectors are particularly preferablyused which can be present in the cell in multiple copies especiallyvectors which can be integrated in a multiple form into the genome ofthe host cell. The invention also concerns combinations of vectors whicheach contain different selection marker genes and which can bepropagated concurrently in a host cell.

[0043] In addition the invention concerns a recombinant cell which istransformed with a nucleic acid according to the invention or a vectoraccording to the invention. The recombinant cell is preferably a yeastcell in particular a methylotrophic yeast cell such as Pichia pastorisor Hansenula polymorpha.

[0044] Finally the invention concerns a recombinant trypsinogen which iscoded by a nucleic acid according to the invention. The recombinanttrypsinogen has a propeptide sequence which is shortened compared to thenatural propeptide sequence and contains an enterokinase recognitionsite. The recombinant trypsinogen according to the invention preferablyhas the amino acid sequence shown in SEQ ID NO. 23.

[0045] The present invention is also elucidated by the following figuresand examples.

[0046]FIG. 1: shows a plasmid map of the expression plasmid pTRYP-9containing the complete recombinant trypsinogen,

[0047]FIG. 2: shows a plasmid map of the expression plasmid pTRYP-11containing the shortened recombinant trypsinogen (sh-trypsinogen) andthe Zeocin resistance marker gene (ZeoR),

[0048]FIG. 3: shows a plasmid map of the expression plasmid pTRYP-13containing the shortened recombinant trypsinogen (sh-trypsinogen) and akanamycin/G418 selection marker gene (KanR).

EXAMPLES Methods

[0049] Recombinant DNA Techniques

[0050] Standard methods were used to manipulate DNA as described bySambrook, J. et al. (1989) in Molecular Cloning: A Laboratory Manual.Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Themolecular biological reagents were used according to the manufacturer'sinstructions.

[0051] Protein Determination

[0052] The protein determination of purified trypsin was carried out bymeasuring the absorbance at 280 mn. A value of 13.6 was used for A1%/280 nm for 1 cm path length.

[0053] Calculation:

Protein [mg/ml]=(10 [mg/ml]*ΔA_(sample)*dilution)/13.6

[0054]Pichia pastoris and Expression Vectors for Pichia pastoris

[0055] The catalogue and instruction manuals from Invitrogen were usedas instructions for handling Pichia pastoris and the expression vectors.The vectors for expressing the shortened recombinant trypsinogen arebased on the vectors pPICZαA and pPIC9K from Invitrogen.

Example 1

[0056] Gene synthesis of the complete recombinant trypsinogen withoptimized codon usage for expression in yeast

[0057] One of the preferred methods for providing the method accordingto the invention is to synthesize a codon-optimized gene sequence. Inorder to optimize each codon for expression in yeast it was necessary tocarry out a complete de novo synthesis of the ca. 700 bp gene whichcodes for the complete recombinant trypsinogen. It was possible tooptimize each codon when necessary by utilizing the degenerate code inthe retranslation of the amino acid sequence of porcine trypsinogenaccording to SEQ ID NO.1. For this purpose the gene was divided into 18oligonucleotides having a length of 54 to 90 nucleotides. Theoligonucleotides were designed as an alternating sequence of sensestrand and counter-strand fragments whose 5′ and 3′ ends each overlappedin a complementary manner with the neighbouring oligonucleotides. Theoverlapping region was selected in each case such that unspecificbinding was largely prevented in the annealing reaction in thesubsequent PCR reaction. The oligonucleotides at the 5′ and 3′ end ofthe gene were provided upstream and downstream of the coding region withrecognition sites for restriction endonucleases which could be used forthe later insertion of the synthetic gene according to SEQ ID NO.2 intoexpression vectors. Thus a recognition site for the restrictionendonuclease EcoRI was incorporated upstream and a recognition site forthe restriction endonuclease XbaI was incorporated downstream. Thesequences of the oligonucleotides are shown in SEQ ID NO.3 to 20.

[0058] Gene synthesis was carried out by means of the PCR reaction. Forthis the coding region was firstly divided into three segments(oligonucleotides 3 to 8, 9 to 14, 15 to 20) and these segments weregenerated in separate PCR reactions using overlapping complementaryoligonucleotides. In this process the gene fragment was extended in astepwise manner till the full length product was formed which was thenamplified in subsequent cycles. The annealing temperature was selectedaccording to the overlapping region with the lowest melting temperature.

[0059] The three segments were subsequently analysed by agarose gelelectrophoresis, the products having the expected length were isolatedfrom the gel by means of the QIAquick Gel Extraction Kit (Qiagen) andsynthesized to form the complete gene product in a further PCR reaction.The first 5 cycles of the PCR reaction were carried out without addingthe primer at the 5′ end and at the 3′ end of the entire gene so that atfirst a few fragments of the gene product of the expected length wereformed from the three segments. The annealing temperature was selectedaccording to the overlapping region with the lowest melting temperature.Then the terminal primers were added and the annealing temperature wasincreased according to the annealing temperature of the primer with thelowest melting temperature. The gene fragment of the expected length wasthen amplified in a further 25 cycles. The PCR fragment was checked bysequencing.

Example 2

[0060] Generation of the Shortened Trypsinogen Gene

[0061] The codons for the first 20 amino acids of the naturallyoccurring trypsinogen were deleted by means of a specially designed 5′primer according to SEQ ID NO. 21 such that only the codons for therecognition sequence of the enteropeptidase enterokinaseAsp-Asp-Asp-Asp-Lys and the codons Glu-Phe necessary for cloning intothe expression vectors for Pichia pastoris due to the EcoRI restrictionendonuclease recognition sequence remain as the sequence of thepropeptide at the N-terminus of the shortened recombinant trypsinogen.The deletion was introduced by a PCR reaction on the PCR fragment of thegene for the complete recombinant trypsinogen using the 5′ primeraccording to SEQ ID NO. 21 and the 3′ primer according to SEQ ID NO. 20.The DNA sequence and the amino acid sequence of the shortenedrecombinant trypsinogen are shown in SEQ ID NO. 22 and SEQ ID NO. 23respectively.

Example 3

[0062] Cloning the PCR Fragments of the Complete Recombinant TrypsinogenGenerated by Gene Synthesis and of the Shortened Recombinant TrypsinogenInto Expression Vectors for Pichia pastoris

[0063] The PCR fragments were recleaved with EcoRI and XbaI (RocheDiagnostics GmbH), isolated again (QIAquick Gel Extraction Kit/Qiagen)and subsequently ligated into a fragment of the expression vectorpPICZαA (Invitrogen) linearized with EcoRI and XbaI (Roche DiagnosticsGmbH) and isolated with the QIAquick Gel Extraction Kit/Qiagen. For this1 μl (20 ng) vector fragment and 3 μl (100 ng) PCR fragment, 1 μl 10×ligase buffer (Sambrook et al., 1989 B.27), 1 μl T4 DNA ligase, 4 μlsterile H₂O_(redistilled) were pipetted, carefully mixed and incubatedovernight at 16° C. In this vector the synthetic gene is under thecontrol of the AOX 1 promoter (promoter for the alcohol oxidase 1 fromPichia pastoris) that can be induced with methanol (Mallinckrodt BakerB. V.) and is located in the correct reading frame behind the signalpeptide of the a factor from Saccharomyces cerevisiae. In order to checkthis and isolate the plasmid, 5 μl of the ligation mixture were thentransformed in 200 μl competent cells (Hanahan (1983) of E. coli XL1Blue(Stratagene). A 30 min incubation on ice was followed by a heat shock(90 sec at 42° C.). Subsequently the cells were transferred to 1 ml LBmedium and incubated for 1 hour at 37° C. in LB medium for phenotypicexpression. Aliquots of this transformation mixture were plated out onLB plates using 100 μg/ml Zeocin as the selection marker and incubatedfor 15 hours at 37° C. The plasmids were isolated from the grown clones(Sambrook, J. et al. (1989) In. Molecular cloning: A Laboratory Manual.Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) and thenchecked for an error-free base sequence by means of restriction analysisand sequencing. The expression vectors formed in this manner whichcontain a synthetic gene for the complete recombinant trypsinogen or ashortened recombinant trypsinogen were named pTRYP-9 (see FIG. 1) andpTRYP-11 (see FIG. 2).

[0064] Transformation of pTRYP-9 and pTRYP-11 Into Pichia pastoris

[0065] For the transformation of pTRYP-9 and pTRYP-11 into Pichiapastoris X-33, GS115 or KM71H with subsequent integration into thegenome, the vector was firstly linearized with SacI (Roche DiagnosticsGmbH). The transformation was carried out by electroporation using theGene Pulser II (Biorad). For this a colony of Pichia pastoris wasinoculated with 5 ml YPD medium (Invitrogen) and incubated overnight at30° C. while shaking. The overnight culture was subsequentlyreinoculated 1:2000 in 200 ml fresh YPD medium (Invitrogen) andincubated overnight at 30° C. while shaking until an OD₆₀₀ of 1.3 to 1.5was reached. The cells were centrifuged (1500×g/5 minutes) and thepellet was resuspended in 200 ml ice-cold, sterile water (0° C.). Thecells were again centrifuged (1500×g/5 minutes) and resuspended in 100ml ice-cold, sterile water (0° C.). The cells were again centrifuged andresuspended in 10 ml ice-cold (0° C.) 1 M sorbitol (ICN). The cells wereagain centrifuged and resuspended in 0.5 ml ice-cold (0° C.) 1 Msorbitol (ICN). The cells isolated in this manner were kept on ice andimmediately used for the transformation.

[0066] 80 μl of the cells were admixed with ca. 1 μg linearized pTRYP-9or pTRYP-11 vector DNA and the entire mixture was transferred into anice-cold (0° C.) electroporation cuvette and incubated for a further 5minutes on ice. Subsequently the cuvette was placed in the Gene PulserII (Biorad) and transformation was carried out at 1 kV, 1 kΩ and 25 μF.After electroporation the mixture was admixed with 1 ml 1 M sorbitol(ICN) and subsequently 100 to 150 μl was plated out on a YPDS agar plate(Invitrogen) containing 100 μg/ml Zeocin (Invitrogen). The plates weresubsequently incubated for 2-4 days at 30° C.

[0067] The clones were reinoculated on raster MD (=minimal dextrose)plates and analysed further. Grown clones were picked, resuspended in 20μl sterile water, lysed (1 hour, 30° C., 10 min, −80° C.) with 17.5 Ulyticase (Roche Diagnostics GmbH) and directly examined for correctintegration of the synthetic trypsinogen expression cassette by means ofPCR.

[0068] Clones which had integrated the complete expression cassetteduring transformation into the genome, were then used in expressionexperiments.

[0069] Expression of the Complete and Shortened Recombinant Trypsinogen

[0070] Positive clones were inoculated in 3 ml BMGY medium (Invitrogen)and incubated overnight at 30° C. while shaking. Subsequently the OD wasdetermined at 600 nm and they were reinoculated in 10 ml BMMY medium(Invitrogen) pH 3.0 such that the resulting OD₆₀₀ was 1. The BMMY medium(Invitrogen) pH 3.0 contains methanol (Mallinckrodt Baker B. V.) whichinduces the expression of the complete or shortened recombinanttrypsinogen via the AOX1 promoter.

[0071] The shaking flasks were incubated at 30° C. while shaking,samples were taken every 24 hours, the OD₆₀₀ was determined, an aliquotwas taken to check the expression of the complete and shortenedrecombinant trypsinogen by means of SDS polyacrylamide gelelectrophoresis and each was supplemented with 0.5% methanol(Mallinckrodt Baker B. V.) for further induction. The expressionexperiments were carried out for 72 hours.

[0072] Analysis of the Expression of the Complete and ShortenedRecombinant Trypsinogen by Means of SDS Gel Electrophoresis

[0073] 500 μl were taken from each expression culture, the OD₆₀₀ wasdetermined and the cells were centrifuged. The culture supernatant wasstored and the cell pellet was resuspended for the lysis in anappropriate amount of Y-PERTM (50 to 300 μl/Pierce) for the OD₆₀₀ andshaken for 1 hour at room temperature. Subsequently the lysate wascentrifuged to separate cell debris (15000×g/5 minutes) and thesupernatant was transferred to fresh reaction vessels. 10 μl lysate and10 μl of the culture supernatant were applied to an SDS polyacrylamidegel and the proteins were separated according to size by applying anelectrical field.

[0074] Surprisingly it was possible to identify weak bands in theculture supernatants of clones containing the complete recombinanttrypsinogen as well as of clones containing the shortened recombinanttrypsinogen that did not occur in the control clones. Control clones areto be understood as Pichia pastoris X33 cells that have been transformedwith the starting vector pPICZαA and that have been grown and inducedanalogously to the expression clones for trypsinogen. The migrationproperties of the new protein bands in the expression clones correspondsto the calculated molecular weight and is slightly higher than that ofbovine trypsin that had been applied as the control marker. This slightdifference in size indicates an intact, non-activated trypsinogen.

[0075] Surprisingly the recombinant shortened trypsinogen was expressedsignificantly more strongly than the recombinant complete trypsinogen.

Example 4

[0076] Increasing the Expression Yield by Multiple Transformation

[0077] The best clones from the expression experiments with therecombinant shortened trypsinogen were again prepared forelectroporation as described above and again transformed with 1 μglinearized pTRYP-11 vector DNA and the transformation mixture was platedout on YPDS agar plates (Invitrogen) containing 1000 to 2000 μg/mlZeocin (Invitrogen). This increases the selection pressure in such a waythat only clones can grow that have integrated several copies of theexpression vector pTRYP-11 into the genome and hence also several copiesof the respective resistance gene (in this case Zeocin®). The Zeocin®resistance protein is the product of the bleomycin gene fromStreptoalloteichus hindustanus (Calmels, T. et al., (1991); Drocourt, D.et al., (1990)), which binds Zeocin® in a stoichiometric concentrationratio and hence makes the cell resistant to Zeocin®. The higher theconcentration of Zeocin® in the YPDS agar plates, the more resistanceprotein has to be produced by the cell in order to quantitatively bindZeocin® and thus allow growth. This is among others possible whenmultiple copies of the resistance gene are integrated into the genome.Clones were reinoculated on raster MD plates as described above.Subsequently these clones were in turn checked for trypsinogenexpression and secretion as described above.

[0078] Surprisingly it was possible after this measure to identifyclones having considerably increased expression yield of the shortenedrecombinant trypsinogen secreted into the culture supernatant after SDSpolyacrylamide gel electrophoresis.

Example 5

[0079] Increasing the Expression Yield by Using a Second SelectionPressure

[0080] Increasing the Zeocin® concentration above 2000 μg/ml did notlead to an improved expression yield of the shortened recombinanttrypsinogen. In order to further increase the gene copy number in theexpression clones of the gene according to SEQ ID NO. 22 which codes forthe recombinant shortened trypsinogen and is codon-optimized forexpression in yeast, additional expression vectors were integrated intothe genome of the expression clones prepared in examples 3 and 4 havingthe highest expression yield by means of a second selection pressure,preferably G418 (Roche Diagnostics GmbH).

[0081] For this purpose a part of the expression cassette from pTRYP-11consisting of a part of the AOX 1 promoter, the gene for the signalpeptide of the α factor of Saccharomyces cerevisiae, the codon-optimizedgene for the recombinant shortened trypsinogen according to SEQ IDNO.22, is cut out with the restriction endonucleases SacI and XbaI frompTRYP-11, the restriction mixture is separated on a 1% agarose gel andthe 1693 bp fragment is isolated from the gel (QIAquick Gel ExtractionKit/Qiagen). At the same time the vector pPIC9K (Invitrogen) was cleavedwith SacI and NotI, the restriction mixture was separated on a 1%agarose gel and the 8956 bp vector fragment was isolated from the gel(QIAquick Gel Extraction Kit/Qiagen). The XbaI overhang of the fragmentfrom pTRYP-11 and the NotI overhang from pPIC9K was filled up withKlenow polymerase (Roche Diagnostics GmbH) to form blunt ends accordingto the manufacturer's instructions. Subsequently the two fragmentsobtained in this manner were ligated as described above. The ligationmixture was transformed in E. coli XL1 Blue (Stratagene) as describedabove (the clones containing plasmid were selected by 100 μg/mlampicillin in the nutrient plates) and checked by means of restrictionanalysis and sequencing. The expression vector formed in this way wasnamed pTRYP-13 (see FIG. 3).

[0082] The integration of the expression vector pTRYP-13 into the genomeof Pichia pastoris was selected by means of G418 (Roche DiagnosticsGmbH).

[0083] The clones having the highest trypsinogen expression yield fromthe multiple transformation with pTRYP-11 (Zeocin resistance) wereprepared for electroporation as described above and transformed with 1μg of the vector fragment from pTRYP-13 (G418 resistance) linearizedwith Sall (Roche Diagnostics GmbH). The transformation mixture wassubsequently stored for 1 to 3 days at 4° C. in 1 M sorbitol (ICN) (forthe formation of G418 resistance), then 100 to 200 μl was plated out onYPD plates (Invitrogen) containing 1, 2 and 4 mg/ml G418 (RocheDiagnostics GmbH) and incubated for 3 to 5 days at 30° C. The clonesresulting therefrom preferably from the YPD plates with the highest G418concentration, were again examined for an increased expression of theshortened recombinant trypsinogen using SDS polyacrylamide gelelectrophoresis as described above.

[0084] After this process is was surprisingly possible to again identifyclones having an increased expression yield of the shortened recombinanttrypsinogen in the culture supernatant after SDS polyacrylamide gelelectrophoresis.

Example 6

[0085] Isolating Trypsinogen from the Culture Supernatant and Activation

[0086] 6.1

[0087] The culture supernatant was separated from the cells bymicrofiltration, centrifugation or filtration. The trypsinogen waspurified by chromatography on phenyl Sepharose fast flow (Pharmacia).The chromatography was carried out in a pH range of 2-4. Autocatalyticactivation was started by rebuffering the pH to 7-8 in the presence of20 mM CaCl₂. This autocatalytic activation can be terminated again bychanging the pH back into the range of 2-4. Active trypsin was purifiedby chromatography on benzamidine Sepharose (e.g. SP-Sepharose®XL, ff)(Pharmacia/package insert) or on an ion exchanger. Trypsin is stored atpH 1.5-3 in order to avoid autolysis. The specific activity of thepurified trypsin is 180-200 U/mg protein.

[0088] 6.2

[0089] The entire fermentation broth is diluted in a ratio of about 1:2to 1:4 with ammonium acetate buffer (5-20 mM) containing 5-30 mM calciumchloride, pH 3.5 and purified by means of an expanded bed chromatography(McCornick (1993); EP 0 699 687) using a cation exchanger (e.g.Streamline®SP, XL). In this case the chromatography is carried out inthe presence of the cells. The cells are simultaneously separated by thechromatography step. Subsequently the procedure is as described inexample 6.1 (rebuffering/activation etc.).

Example 7

[0090] Activity Determination

[0091] The activity of trypsin was determined using Chromozym TRY(Pentapharn Ltd) in 100 mM Tris pH 8.0, 20 mM CaCl₂ at 25° C. Thephotometric measurement is carried out at 405 nm.

[0092] Abbreviations:

[0093] YPD: yeast peptone dextrose

[0094] YPDS: yeast peptone dextrose sorbitol

[0095] BMGY: buffered glycerol-complex medium

[0096] BMMY: buffered methanol-complex medium

[0097] SDS: sodium dodecyl sulfate

[0098] List of Literature References:

[0099] Bricteuz-Gregoire, Schyns R., Florkin M (1966)

[0100] Biochim. Biophys. Acta 127: pp 277

[0101] Calmels T., Parriche M., Durand H., Tiraby G. (1991),

[0102] Curr. Genet. 20: pp 309

[0103] Charles M., Rovery M., Guidoni A., Desnuelle P. (1963)

[0104] Biochim. Biophys. Acta 69: pp 115-129

[0105] Desnuelle P. (1959)

[0106] The Enzymes 2^(nd) Edition vol 4 Editor Boyer, Acad. PressNY.pp.119

[0107] Drocourt, D., Calmels T., Reynes J. P., Baron M., Tiraby G.(1990),

[0108] Nucleic Acid Research 18: pp 4009

[0109] Graf L., Craik C. S., Patthy A., Roczniak S., Fletterick R J.,Rutter W. J. (1987)

[0110] Biochem. 26: pp. 2616

[0111] Graf L., Jancso A., Szilagyi L., Hegyi G., Pinter K., Naray-SzaboG., Hepp J.,

[0112] Mehzihradszky K., Rutter W. J. (1988)

[0113] Proc. Natl. Acad. Sci USA 85, pp 4961

[0114] Hanahan (1983)

[0115] J. Mol. Biol., 166: pp 557

[0116] Higaki J. N., Evnin L. B., Craik C. S. (1989)

[0117] Biochem. 28: pp 9256

[0118] Hedstrom L., Szilagyi L., Rutter W. J. (1992)

[0119] Science 255: pp 1249

[0120] Jurasek L., Fackre D. Smillie L. B. (1969)

[0121] Biochem. Biophys. Res. Commun 37: pp. 99

[0122] Keil B. (1971)

[0123] The Enzymes Vol II, 3^(rd) Edition, Editor Boyer, Acad. PressN.Y. pp 249-275

[0124] Light A., Savithari H. S., Liepnieks J. J. (1980)

[0125] Analytical Biochemistry 106: pp 199-206

[0126] McComick, D. K., Bio/Technol. 11 (1993), 1059; Expanded BedAbsorption, Principles and Methods, Amersham Pharmacia Biotech, EditionAB, ISBN 91-630-5519-8;

[0127] Morihara K. and Tszzuki J. (1969)

[0128] Arch Biochem Biophys 126: pp 971

[0129] Northrop J. H., Kunitz M., Herriott R. (1948)

[0130] Crystalline Enzymes, 2^(nd) Edition, Columbia Univ. Press NY

[0131] Ryan C. A. (1965)

[0132] Arch Biochem Biophys 110: pp 169

[0133] Sambrook, J., Fritsch E. F., Maniatis T. (1989)

[0134] In. Molecular Cloning: A Laboratory Manual. Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.

[0135] Travis J. (1968)

[0136] Biochem Biophys Res Commun. 30: pp 730

[0137] Travis J. and Roberts R. C. (1969)

[0138] Biochemistry 8: pp 2884

[0139] Trop M. and Birk Y. (1968)

[0140] Biochem. J. 109: pp 475

[0141] Wählby S. and Engström L. (1968)

[0142] Biocheim. Biophys. Acta 151: pp 402

[0143] Vasquez, J. R. Evnin L. B., Higaki J. N. Craik C. S. (1989)

[0144] J. Cell. Biochem. 39: pp. 265

[0145] Wählby S. (1968)

[0146] Biochim. Biophys. Acta 151: pp 394

[0147] Willett, W. S., Gillmor S. A., Perona J. J., Fletterick R. J.,Craik C. S. (1995)

[0148] Biochem. 34: pp 2172

[0149] Yee, L. and Blanch, H. W., (1993)

[0150] Biotechnol. Bioeng. 41: pp. 781-790

[0151] WO 97/00316 (NOVO NORDISK AS/Wöldike Helle, Kjeldsen Thomas)

[0152] A PROCESS FOR PRODUCING TRYPSIN

[0153] WO 99/10503 (ROCHE DIAGNOSTICS/Kopetzki Ehrhard, HopfnerKarl-Peter, Bode Wolfram, Huber Robert)

[0154] ZYMOGENIC PROTEASE PRECURSOR THAT CAN BE AUTOCATALYTICALLACTIVATED AND THEIR USE.

[0155] WO 00/17332 (ELI LILLY AND COMPANY/Hanquier Jose Michael,Hershberger Charles Lee, Desplancq Dominique, Larson Jeffrey)

[0156] PRODUCTION OF SOLUBLE RECOMBINANT TRYPSINOGEN ANALOGS WO 01/55429(POLYMUN SCIENTIFIC IMMUBIOLOGISCHE FORSCHUNG GMBH/Mattanovich Diethard,Katinger Hermann, Hohenblum Hubertus, Naschberger Stefan, Weik Robert)

[0157] METHOD FOR THE MANUFACTURE OF RECOMBINANT TRYPSIN

[0158] EP 0 597 681 (ELI LILLY AND COMPANY/Greaney Michael Gerard,Rostech Paul Robert)

[0159] EXPRESSION VECTORS FOR THE BOVINE TRYPSIN AND TRYPSINOGEN ANDHOST CELLS TRANSFORMED THEREWITH

[0160] EP 0 699 687 (MITSUBISHI PHARMA CORPORATION/Noda Munehiro, SumiAkinori, Ohmura Takao, Yokoyama Kazmasa)

[0161] PROCESS FOR PURIFYING RECOMBINANT HUMAN SERUM ALBUMIN

1 23 1 247 PRT Porcine 1 Ile Pro Asn Thr Phe Val Leu Leu Ala Leu Leu GlyAla Ala Val Ala 1 5 10 15 Phe Pro Thr Asp Asp Asp Asp Lys Ile Val GlyGly Tyr Thr Cys Ala 20 25 30 Ala Asn Ser Ile Pro Tyr Gln Val Ser Leu AsnSer Gly Ser His Phe 35 40 45 Cys Gly Gly Ser Leu Ile Asn Ser Gln Trp ValVal Ser Ala Ala His 50 55 60 Cys Tyr Lys Ser Arg Ile Gln Val Arg Leu GlyGlu His Asn Ile Asp 65 70 75 80 Val Leu Glu Gly Asn Glu Gln Phe Ile AsnAla Ala Lys Ile Ile Thr 85 90 95 His Pro Asn Phe Asn Gly Asn Thr Leu AspAsn Asp Ile Met Leu Ile 100 105 110 Lys Leu Ser Ser Pro Ala Thr Leu AsnSer Arg Val Ala Thr Val Ser 115 120 125 Leu Pro Arg Ser Cys Ala Ala AlaGly Thr Glu Cys Leu Ile Ser Gly 130 135 140 Trp Gly Asn Thr Lys Ser SerGly Ser Ser Tyr Pro Ser Leu Leu Gln 145 150 155 160 Cys Leu Lys Ala ProVal Leu Ser Asp Ser Ser Cys Lys Ser Ser Tyr 165 170 175 Pro Gly Gln IleThr Gly Asn Met Ile Cys Val Gly Phe Leu Glu Gly 180 185 190 Gly Lys AspSer Cys Gln Gly Asp Ser Gly Gly Pro Val Val Cys Asn 195 200 205 Gly GlnLeu Gln Gly Ile Val Ser Trp Gly Tyr Gly Cys Ala Gln Lys 210 215 220 AsnLys Pro Gly Val Tyr Thr Lys Val Cys Asn Tyr Val Asn Trp Ile 225 230 235240 Gln Gln Thr Ile Ala Ala Asn 245 2 744 DNA Artificial sequenceSynthetic gene 2 attccaaata cttttgtttt gttggctttg ttgggtgctg ctgttgcttttccaactgat 60 gatgatgata aaattgttgg tggttatact tgtgctgcta attctattccatatcaagtt 120 tctttaaatt ctggttctca tttttgtggt ggttctttga ttaattctcaatgggttgtt 180 tctgctgctc attgttacaa atcaagaatc caagttagat tgggtgaacataatattgat 240 gttttggaag gtaatgaaca atttattaat gctgctaaaa ttattactcatccaaatttt 300 aatggtaata ctttggataa tgatattatg ttgattaaat tgtcttctccagctacttta 360 aattcaagag ttgctactgt ttctttgcca agatcttgtg ctgctgctggtactgaatgt 420 ttgatttctg gttggggtaa tactaaatct tctggttctt cttatccatctttgttgcaa 480 tgtttgaaag ctccagtttt gtctgattct tcttgtaaat cttcttacccaggtcaaatt 540 actggtaata tgatttgtgt tggttttttg gaaggtggta aagattcttgtcaaggtgat 600 tctggtggtc cagttgtttg taatggtcaa ttgcaaggta ttgtttcttggggttatggt 660 tgtgctcaaa aaaataaacc aggtgtttac actaaagttt gtaattatgttaattggatt 720 caacaaacta ttgctgctaa ttag 744 3 56 DNA Artificialsequence Primer 3 gcggaattca ttccaaatac ttttgttttg ttggctttgt tgggtgctgctgttgc 56 4 54 DNA Artificial sequence Primer 4 ccaccaacaa ttttatcatcatcatcagtt ggaaaagcaa cagcagcacc caac 54 5 62 DNA Artificial sequencePrimer 5 gatgataaaa ttgttggtgg ttatacttgt gctgctaatt ctattccatatcaagtttct 60 tt 62 6 64 DNA Artificial sequence Primer 6 gaattaatcaaagaaccacc acaaaaatga gaaccagaat ttaaagaaac ttgatatgga 60 atag 64 7 64DNA Artificial sequence Primer 7 ggtggttctt tgattaattc tcaatgggttgtttctgctg ctcattgtta caaatcaaga 60 atcc 64 8 62 DNA Artificial sequencePrimer 8 ccttccaaaa catcaatatt atgttcaccc aatctaactt ggattcttgatttgtaacaa 60 tg 62 9 63 DNA Artificial sequence Primer 9 taatattgatgttttggaag gtaatgaaca atttattaat gctgctaaaa ttattactca 60 tcc 63 10 62DNA Artificial sequence Primer 10 caacataata tcattatcca aagtattaccattaaaattt ggatgagtaa taattttagc 60 ga 62 11 64 DNA Artificial sequencePrimer 11 ctttggataa tgatattatg ttgattaaat tgtcttctcc agctactttaaattcaagag 60 ttgc 64 12 58 DNA Artificial sequence Primer 12 ccagcagcagcacaagatct tggcaaagaa acagtagcaa ctcttgaatt taaagtag 58 13 61 DNAArtificial sequence Primer 13 cttgtgctgc tgctggtact gaatgtttgatttctggttg gggtaatact aaatcttctg 60 g 61 14 58 DNA Artificial sequencePrimer 14 gagctttcaa acattgcaac aaagatggat aagaagaacc agaagatttagtattacc 58 15 60 DNA Artificial sequence Primer 15 gttgcaatgtttgaaagctc cagttttgtc tgattcttct tgtaaatctt cttacccagg 60 16 59 DNAArtificial sequence Primer 16 ccaaaaaacc aacacaaatc atattaccagtaatttgacc tgggtaagaa gatttacaa 59 17 60 DNA Artificial sequence Primer17 gatttgtgtt ggttttttgg aaggtggtaa agattcttgt caaggtgatt ctggtggtcc 6018 57 DNA Artificial sequence Primer 18 ccaagaaaca ataccttgca attgaccattacaaacaact ggaccaccag aatcacc 57 19 51 DNA Artificial sequence Primer 19gcaaggtatt gtttcttggg gttatggttg tgctcaaaaa aataaaccag g 51 20 90 DNAArtificial sequence Primer 20 gcgtctagac taattagcag caatagtttgttgaatccaa ttaacataat tacaaacttt 60 agtgtaaaca cctggtttat ttttttgagc 9021 48 DNA Artificial sequence Primer 21 ccgggaattc gatgatgatg ataaaattgttggtggttat acttgtgc 48 22 687 DNA Artificial sequence Synthetic gene 22gatgatgatg ataaaattgt tggtggttat acttgtgctg ctaattctat tccatatcaa 60gtttctttaa attctggttc tcatttttgt ggtggttctt tgattaattc tcaatgggtt 120gtttctgctg ctcattgtta caaatcaaga atccaagtta gattgggtga acataatatt 180gatgttttgg aaggtaatga acaatttatt aatgctgcta aaattattac tcatccaaat 240tttaatggta atactttgga taatgatatt atgttgatta aattgtcttc tccagctact 300ttaaattcaa gagttgctac tgtttctttg ccaagatctt gtgctgctgc tggtactgaa 360tgtttgattt ctggttgggg taatactaaa tcttctggtt cttcttatcc atctttgttg 420caatgtttga aagctccagt tttgtctgat tcttcttgta aatcttctta cccaggtcaa 480attactggta atatgatttg tgttggtttt ttggaaggtg gtaaagattc ttgtcaaggt 540gattctggtg gtccagttgt ttgtaatggt caattgcaag gtattgtttc ttggggttat 600ggttgtgctc aaaaaaataa accaggtgtt tacactaaag tttgtaatta tgttaattgg 660attcaacaaa ctattgctgc taattag 687 23 228 PRT Artificial sequenceShortened Trypsinogen 23 Asp Asp Asp Asp Lys Ile Val Gly Gly Tyr Thr CysAla Ala Asn Ser 1 5 10 15 Ile Pro Tyr Gln Val Ser Leu Asn Ser Gly SerHis Phe Cys Gly Gly 20 25 30 Ser Leu Ile Asn Ser Gln Trp Val Val Ser AlaAla His Cys Tyr Lys 35 40 45 Ser Arg Ile Gln Val Arg Leu Gly Glu His AsnIle Asp Val Leu Glu 50 55 60 Gly Asn Glu Gln Phe Ile Asn Ala Ala Lys IleIle Thr His Pro Asn 65 70 75 80 Phe Asn Gly Asn Thr Leu Asp Asn Asp IleMet Leu Ile Lys Leu Ser 85 90 95 Ser Pro Ala Thr Leu Asn Ser Arg Val AlaThr Val Ser Leu Pro Arg 100 105 110 Ser Cys Ala Ala Ala Gly Thr Glu CysLeu Ile Ser Gly Trp Gly Asn 115 120 125 Thr Lys Ser Ser Gly Ser Ser TyrPro Ser Leu Leu Gln Cys Leu Lys 130 135 140 Ala Pro Val Leu Ser Asp SerSer Cys Lys Ser Ser Tyr Pro Gly Gln 145 150 155 160 Ile Thr Gly Asn MetIle Cys Val Gly Phe Leu Glu Gly Gly Lys Asp 165 170 175 Ser Cys Gln GlyAsp Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu 180 185 190 Gln Gly IleVal Ser Trp Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro 195 200 205 Gly ValTyr Thr Lys Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr 210 215 220 IleAla Ala Asn 225

1. A method for the recombinant production of trypsin comprising thesteps: a) transforming a host cell with a recombinant nucleic acid whichcodes for a trypsinogen with an enterokinase recognition site in thepropeptide sequence in a form that can be secreted by the host cell, b)culturing the host cell under conditions which enable an expression ofthe recombinant nucleic acid and a secretion of the expression productinto the culture medium, the conditions being selected such that anautocatalytic cleavage of the propeptide sequence is substantiallyprevented, c) isolating the expression product from the culture medium,d) cleaving the propeptide sequence to form active trypsin and e)optionally separating non-cleaved trypsinogen molecules from activetrypsin.
 2. A method as claimed in claim 1, wherein a yeast cell is usedas the host cell.
 3. A method as claimed in claim 1, wherein Pichiapastoris or Hansenula polymorpha is used as the host cell.
 4. A methodas claimed in claim 1, wherein a recombinant nucleic acid is used whichcodes for a trypsinogen fused to a signal peptide that mediatessecretion.
 5. A method as claimed in claim 1, wherein a recombinantnucleic acid with an optimized codon usage for the host cell is used. 6.A method as claimed in claim 1, wherein a recombinant nucleic acid isused which codes for a trypsinogen having a shortened propeptidesequence.
 7. A method as claimed in claim 1, wherein a recombinantnucleic acid is used which codes for a porcine trypsinogen.
 8. A methodas claimed in claim 1, wherein a recombinant nucleic acid is used havingthe nucleotide sequence shown in SEQ ID NO.
 22. 9. A method as claimedin claim 1, wherein the recombinant nucleic acid is expressed underacidic conditions in particular at pH 3-4.
 10. A method as claimed inclaim 9, wherein the host cell is cultured before expression of therecombinant nucleic acid under conditions that are essentially optimalfor the growth of the host cell.
 11. A method as claimed in claim 10,wherein the host cell is cultured before expression of the recombinantnucleic acid at pH 5-7 up to a predetermined optical density and thenthe expression of the recombinant nucleic acid is induced.
 12. A methodas claimed in claim 1, wherein the propeptide sequence is cleaved byaddition of recombinant trypsin or/and by autocatalytic cleavage.
 13. Amethod as claimed in claim 1, wherein the culture medium containing thehost cells is admixed with a buffer containing calcium ions andsubsequently the steps (c), (d) and optionally (e) are carried out. 14.A nucleic acid comprising a recombinant nucleic acid that codes for atrypsinogen having an enterokinase recognition site in the propeptidesequence, wherein the propeptide sequence is shortened compared to thenatural propeptide sequence.
 15. A nucleic acid as claimed in claim 14,wherein the propeptide sequence consists of an enterokinase recognitionsite having the amino acid sequence Asp-Asp-Asp-Asp-Lys and optionallyup to 5 additional amino acids located N-terminal thereof.
 16. A nucleicacid as claimed in claim 14, wherein the nucleic acid comprises thenucleotide sequence shown in SEQ ID NO.
 22. 17. A nucleic acid asclaimed in claim 14, wherein the nucleic acid is in operative linkagewith a regulatable expression control sequence.
 18. A recombinant vectorcomprising at least one copy of a recombinant nucleic acid as claimed inone of the claims 14 to
 17. 19. A recombinant host cell, wherein thecell is transformed with a nucleic acid as claimed in one of the claims14 to 17 or with a vector as claimed in claim
 18. 20. A recombinanttrypsinogen comprising a trypsin sequence and a propeptide sequencewhich is shortened compared to the natural propeptide sequence andcontains an enterokinase recognition site.
 21. Trypsinogen as claimed inclaim 20, wherein the propeptide sequence consists of an enterokinaserecognition site having the amino acid sequence Asp-Asp-Asp-Asp-Lys andoptionally up to 5 additional amino acids located N-terminal thereof.22. Trypsinogen as claimed in claim 20, wherein the trypsinogen has theamino acid sequence shown in SEQ ID NO. 23.